Ric-8A and Ric-8B are positive regulators of heterotrimeric G protein a subunit function. We have recently defined the cellular action of Ric-8 proteins towards Ga subunits. Ric-8 proteins act as molecular chaperones of nucleotide-free Ga conformations during biosynthetic Ga protein folding. In cells in which Ric-8 genes are deleted, cellular abundances of subsets of Ga subunits were reduced by 95%. Ric-8 proteins also exhibit in vitro guanine nucleotide exchange stimulatory activity towards Ga subunits, and we now believe this to be an in vitro phenomenon stemming from the capacity of Ric-8 to induce a partially folded Ga state that has reduced affinity for GDP. We will develop our hypothesis that Ric-8 proteins are Ga subunit molecular chaperones and investigate the mechanisms by which Ric-8 proteins work with other cellular chaperones including the TRiC/CCT (Chaperonin) complex and HSC70/90 systems to fold individual Ga subunits. We have preliminary evidence that there are complex rules and unique chaperone requirements of individual Ga subunits. We will then test a hypothesis that mouse tissue-specific Ric-8A gene deletion can genetically suppress oncogenic G protein or overactive GPCR-driven cancers. Two mouse melanoma models will be used in which GNAQ/11-Q209L (constitutively-active) alleles are the oncogenic driver mutations of metastatic ocular melanoma, and ectopic mGluR5 expression from a melanocyte promoter elicited robust melanoma. We hypothesize that melanocyte mGluR5 inappropriately activates Gq/11 to promote melanoma. Upon proof-of-concept that Ric-8 deletion will suppress the actions of these oncogenes by reducing Gaq/11 cellular abundances, we will perform screens to identify small molecule inhibitors of Ric-8 and G protein interactions. Our goal is to develop Ric-8 inhibitors as a means to indirectly block G protein disease signaling by reducing the abundance of (mutant) Ga subunits folded by Ric-8. Ric-8 inhibitors may prove efficacious against oncogenic G protein diseases and/or as augmentation therapies for existing GPCR therapeutics. We will also conduct a small project to investigate Ric-8A regulation by phosphorylation events and 14-3-3 binding to Ric-8A.